DESCRIPTION: Recent studies indicate apoptosis is an integral mechanism of ischemic brain damage and is particularly relevant in the ischemic penumbra area where neurons are challenged by hypoxia, but receive residual blood supply. The ultimate goal of this project is to identify the signal transduction mechanisms leading to Caspase activation and apoptosis in cerebral ischemia. With this in mind, a mouse model of global based on unilateral common carotid artery occlusion followed by hypoxia was created. Four major findings are presented in the preliminary experiments. First, the combined stress of ischemia and hypoxia triggers the activation of Caspase-3 causing apoptosis of hippocampal neurons. Second, the hippocampal damage is associated with activation of the c-Jun NH2-terminal kinases (JNK) signaling pathway. Third, deletion of the neural-and heart-specific Jnk3 isoform protects neurons from ischemic apoptosis. Finally, the apoptotic neurons in cerebral ischemia-hypoxia progress into the S-phase of cell cycle and incorporate bromodeoxyuridine (BrdU). These results suggest: (1). The neural-and heart-specific JNK3 signaling pathway plays a pivotal role in ischemic apoptosis. (2) the downstream mechanisms may involve aberrant cell cycle checkpoints, transcriptional induction of mitochondrial release of death-promoting factors. Aim 1: To identify which JNK isoform is important for apoptosis induced by ischemia-hypoxia and whether the inhibition of JNK3 signaling protects against cerebral infarction. We will test the role of the constitutively active JNK1 isoform in global ischemia and the stress-induced JNK3 in focal ischemia. Aim 2: To determine the mechanism by which post-mitotic neurons re-enter the cell cycle in cerebral ischemia and whether the aberrant cell cycle progression is caused by JNK signaling and/or important for neuronal apoptosis. The cellular mechanisms that may abolish the G1/S-phase transition block in cerebral ischemia will be examined. In addition, the functional significance and cause of the aberrant cell cycle progression will be assessed. Aim 3: To elucidate the transcriptional and mitochondrial targets of JNK signaling that promotes neuronal apoptosis in cerebral ischemia. The cDNA microarray technology will be used in an in-vitro neuron culture system to identify the downstream death-promoting genes of JNK signaling. An in-vitro Caspase-3 cleavage assay will be used to test whether the absence of JNK3 attenuates the release of Cytochrome c from mitochondria in neurons under hypoxia and neurotoxin stress. Finally, we will use tw0-dimensional protein electrophoresis of mass spectrometry to identify the mitochondrial targets of JNK signaling.